Myocardial infarction and subsequent congestive heart failure are among the most challenging and prevalent health problems. Stem cell transplantation has the potential to allow cardiac repair, and mesenchymal stem cells (MSC) are among the promising cell types for cardiac regeneration, although many challenges remain, before stem cells will lead to curative therapy, and it is currently more realistic to aim for stem cell-mediated improved revascularization than for cardiomyocyte replacement. One possible approach is to instruct MSC in vitro in ways which will increase their propensity for homing and engraftment to the diseased heart. We hypothesize that the anaphylatoxins C3a and C5a are one such means. Our preliminary studies surprisingly showed that the C3a receptor (C3aR) is translocated to the nucleus in MSC, but not in other cell types. Interestingly, nuclear translocation of other G-protein-coupled receptors has been associated with prolonged activation of signaling pathways and transcriptional activation, which may result in cardioprotection, and indeed MSCs stimulated with C3a up-regulate several trophic and angiogenic factors. Aim 1: To determine the effect of C3a and C5a on MSC functions which are relevant to their cardiac repair potential: Hypothesis: C3a and C5a have effects on MSCs that will improve their potential as transplantable cells for cardiac repair. This is based on the observation that C3a and C5a both cause long-lasting activation of signaling pathways (ERK and Akt) that are associated with MSC expansion, production of cardioprotective growth factors and differentiation toward cardiomyocytes. Important facets of MSC function (cell migration, production of trophic factors, proliferation and differentiation) will be determined in MSCs stimulated with C3a or C5a. These functions will be determined both in vitro and in vivo. Aim 2: To characterize the nuclear translocation of the C3aR and associated signaling molecules in C3a-stimulated MSCs and to determine the effect of the nuclear translocation and the prolonged ERK1/2 and Akt activation on MSC function. Aim 2.1: Hypothesis: The C3aR in MSCs translocates to the nucleus, which is associated with prolonged activation of signaling pathways and transcriptional activation. Nuclear translocation of the C3aR, phospho-ERK1/2, phospho-Akt and beta-arrestin will be determined in nuclear extracts of C3a-stimulated human MSCs. C5a will be used for comparison. Means of inhibiting nuclear translocation of the C3aR will be used to determine the role of the nuclear translocation on cell signaling (Aim 2.1) and cell function (Aim 2.2). Aim 2.2: To determine the effect of the nuclear translocation and the prolonged C3a and C5a-mediated ERK1/2 and Akt activation on MSC function. Hypothesis: The prolonged activation of ERK1/2 and/or Akt pathways - and in the case of the C3aR, the nuclear translocation of this receptor - are responsible for the functional effects of C3a and C5a. The effect of inhibition of ERK1/2 and Akt and of nuclear translocation of the C3aR on specific C3a or C5a mediated functions will be assessed in order to determine which signaling pathway mediates which effect. PUBLIC HEALTH RELEVANCE: Myocardial infarction and subsequent congestive heart failure remain among the most challenging and prevalent health problems. Stem cell transplantation has the potential to allow cardiac repair, and mesenchymal stem cells (MSCs) are among the promising cell types for cardiac regeneration. One possible approach is to instruct MSCs in vitro in ways which will increase their potential for engraftment and homing to the diseased heart. We hypothesize that the chemotactic factors C3a and C5a are one such means. In this proposal the functional role of C3a and C5a on MSC will be investigated.